The present invention broadly relates to the treatment, diagnosis, and prophylactic prevention of Alzheimer""s disease. More specifically, the present invention relates to methods and compositions for preventing the endocytosis and cellular internalization of integral membrane amyloid xcex2-precursor protein (APP) and its subsequent catabolism by blocking or interfering with the association or binding of APP with members of the low density lipoprotein receptor family.
Alzheimer""s disease (AD) is a late onset neurodegenerative disorder characterized by the extracellular deposition of insoluble aggregates composed of the 40 to 42 amino acid Axcex2 peptide in the brain (Glenner and Wong, Biochem. Biophys. Res. Commun. 120:885-890 (1984); Masters et al., EMBO J. 4:2757-2763 (1985)). Axcex2 peptide is derived from an integral membrane protein termed amyloid xcex2-protein precursor protein (APP) (Tanzi et al., Science 235:880 (1987); Kang et al., Nature 325:733-736 (1987)). The function and metabolism of APP have been the subject of intensive study due to the fact that mutations in APP are associated with an autosomal dominant form of AD, (Goate et al., Nature 349:704-707 (1991)) and over-production of APP is the presumptive cause of AD in trisomy 21 (Tanzi et al., Science 235:880 (1987); Hyman et al., Proc. Natl. Acad. Sci. USA 92:3586-3590 (1995)). Multiple APP isoforms can be generated by alternatively splicing of mRNAs. The major isoforms in brain are APP695, APP751, and APP770 containing 695, 751 and 770 amino acids, respectively. These isoforms are transmembranous proteins having large extracellular regions, with hydrophobic membrane spanning domains and short cytoplasmic segments. APP is also a member of an evolutionary conserved family of proteins which include the APP-like proteins, APLP1 and APLP2 (Wasco et al., Proc. Natl. Acad. Sci. USA 89:10758-10762 (1992); Wasco et al., Nature Genet. 5:95-100 (1993); Slunt et al., J. Biol Chem. 269:2637-2644 (1994)).
Secreted forms of APP are generated by proteolytic cleavages within their extracellular domain close to the transmembrane region. The extracellular regions of APP751, APP770, and APLP2 each contain a Kunitz protease inhibitor (KPI) domain encoded by an alternatively-transcribed exon (Kitaguchi et al., Nature 331:530-532 (1988); Tanzi et al., Nature 331:528-530 (1988); Wasco et al., Nature Genet. 5:95-100 (1993); Slunt et al., J. Biol. Chem. 269:2637-2644 (1994)). Secreted forms of APP having the KPI domain correspond to a protease inhibitor that has been identified separately and named protease nexin II (APP/PN-2) (Van Nostrand and Cunningham, J. Biol. Chem. 262:8508-8514 (1987); Oltersdorf et al., Nature 341:144-147 (1989); Van Nostrand et al., Nature 341:546-549 (1989)), a potent inhibitor of the blood coagulation factors IXa (Schmaier et al., J. Clin. Invest. 92:2540-2545 (1993)) and XIa (Van Nostrand et al., J. Biol. Chem. 265:9591-9594 (1990)). APP/PN-2 binds with high affinity to cultured fibroblasts (Johnson-Wood et al., Biochem. Biophys. Res. Commun. 200:1685-1692 (1994)), and APP/PN-2:proteinase complexes are internalized and degraded by cultured cells (Knauer and Cunningham, Proc. Natl. Acad. Sc. USA 79:2310-2314 (1982); Knauer et al., J. Cell. Physiol. 117:385-396 (1983)) although the mechanism for this process is unknown. Recent studies have identified the low density lipoprotein receptor-related protein (LRP) as the receptor responsible for the catabolism of another Kunitz-type inhibitor, tissue factor pathway inhibitor (TFPI) (Warshawsky et al., Proc. Natl. Acad. Sci. USA 91:6664-6668 (1994)).
LRP is a large multiligand receptor (Krieger and Herz, Annu. Rev. Biochem. 63:601-637 (1994)) that is a member of the LDL receptor family, which also includes the LDL receptor (Yamamoto et al., Cell 39:27-38 (1984)), the VLDL receptor (Takahashi et al., Proc. Natl. Acad. Sci. USA 89:9252-9256 (1992)), and glycoprotein 330 (Saito et al., Proc. Natl. Acad. Sci. USA 91:9725-9729 (1994)). A 39 kDa protein, termed the receptor associated protein (RAP) (Strickland et al., J. Biol. Chem. 266:13364-13369 (1991)) binds to members of the LDL receptor family (Williams et al., J. Biol. Chem. 267:9035-9040 (1992); Kounnas et al., J. Biol. Chem. 267:21162-21166 (1992); Battey et al., J. Biol. Chem. 269:23268-23273 (1994)) and blocks their ligand binding capacity. LRP mediates the cellular uptake and subsequent degradation of proteinases, such as tissue-type plasminogen activator (Bu et al., Proc. Natl. Acad. Sci. USA 89:7427-7431 (1992)) and urokinase-type plasminogen activator (Kounnas et al., J. Biol. Chem. 268:21862-21867 (1993)), proteinase-inhibitor complexes, such as xcex12-macroglobulin-proteinase complexes (Ashcom et al., J. Cell Biol. 110: 1041-1048 (1990); Moestrup and Gliemann, J. Biol. Chem. 264:15574-15577 (1989)), serpin-proteinase complexes (Orth et al., Proc. Natl. Acad. Sci. USA 89:7422-7426 (1992); Nykjaer et al., J. Bio. Chem. 267:14543-14546 (1992); Poller et al., J. Biol. Chem. 270:2841-2845 (1995)), matrix proteins, such as thrombospondin (Mikhailenko et al., J. Biol. Chem. 270:9543-9549 (1995)), apolipoprotein E (apoE)-enriched lipoproteins (Kowal et al., J. Biol. Chem. 265:10771-10779 (1990); Beisiegel et al., Nature 341:162-164 (1989)), hepatic lipase (Kounnas et al., J. Biol. Chem. 270:9307-9312 (1995)) and lipoprotein lipase (Chappell et al., J. Biol. Chem. 268:14168-14175 (1993)).
LRP is expressed in many tissues and is a major apoe receptor in the central nervous system (Rebeck et al., Neuron 11:575-580 (1993)). Genetic data implicate inheritance of the xcex54 allele of apoe as a risk factor in AD (Strittmatter et al., Proc. Natl. Acad. Sci. USA 90:1977-1981 (1993); Rebeck et al., Neuron 11:575-580 (1993); Poirier et al., Lancet 342:697-699 (1993); Saunders et al., Neurology 43:1467-1472 (1993)). A possible involvement of LRP in AD is suggested in part by the observation that LRP, as well as apoE and other LRP ligands, decorate senile plaques (Rebeck et al., Ann. Neurol. 37:211-217 (1995)).
The ability of LRP to mediate the cellular catabolism of TFPI, a KPI-containing protein, led to the investigation of the role of LRP in the catabolism of APPs770. The present inventors have found that LRP is capable of binding and mediating the internalization and degradation of APPs770 as well as its complexes with proteinases.
Because catabolism of APP has been shown to generate the Axcex2 peptide, which is believed to be the causative agent of Alzheimer""s Disease, there is a need for compositions and methods which reduce the interaction, cellular internalization and subsequent catabolism of APP.
Accordingly, it is an object of the present invention to provide agents which bind to APP or LDL-receptor family members and reduce the interaction, cellular internalization, and subsequent catabolism of APP. Other objects, features and advantages of the present invention will be set forth in the detailed description of preferred embodiments that follows, and in part will be apparent from the description or may be learned by practice of the invention. These objects and advantages of the invention will be realized and attained by the compositions methods particularly pointed out in the written description and claims hereof.
A first embodiment of the present invention therefore relates to agents which bind to the APP-binding site on the LRP particle (Group I agents) and agents which bind to the LRP-binding site found on APP (Group II agents).
An additional embodiment of the present invention relates to DNA molecules which encode peptides and antibodies that are Group I agents and/or Group II agents and to host organisms that have been transformed with the DNA molecules of the present invention.
The present invention also relates to processes for preparing DNA molecules which encode a functional derivative or a fragment of LRP, RAP or APP. These processes can yield DNA sequences which are inserted into a vector DNA containing expression control sequences in such a way that the expression control sequences regulate the expression of the inserted DNA.
A further embodiment of the present invention relates to methods for preparing polypeptides that are functional derivatives of LRP which comprise taking the polypeptide from the native receptor molecule by enzymatic, such as proteolytic, or chemical, such as reductive, treatment.
An additional embodiment of the invention relates to a process for preparing Group I agents which are a functional derivative of APP or RAP which comprise expressing a recombinant DNA molecule according to the invention.
The present invention further relates to Group II agents which are antibodies, or an antibody fragment containing the antigen binding domain, that bind to the LRP binding site found on APP.
Another embodiment of the present invention relates to processes for preparing Group II agents which are a functional derivative of LRP which comprise expressing a recombinant DNA molecule according to the invention.
The present invention additionally relates to hybrid cell lines that secrete monoclonal antibodies against the LDL-receptor protein which interfere with APP attachment to the LRP receptor.
An additional embodiment of the present invention includes the use of Group I agents and/or Group II agents for qualitatively and/or quantitatively determining or purifying the presence of LRP which is found in a sample.
A further embodiment of the present invention includes a test kit for determining whether a polypeptide is a Group I agent and/or a Group II agent, this kit comprising a carrier means having in close confinement therein one or more container means at least one of which contains an antibody that binds to the LRP binding site found on APP.
Another embodiment of the present invention relates to processes for preparing antibodies that bind to the LRP binding site found on APP, in which a host animal is immunized with one or more polypeptides of Group I and/or Group II, the B-lymphocytes of these host animals are fused with myeloma cells, and a hybrid cell line secreting the monoclonal antibody is subcloned and cultivated.
An additional embodiment of the present invention relates to the use of Group I agents and/or Group II agents, or the native receptor molecules of the LDL-receptor family or pharmaceutically suitable salts thereof, for the therapeutic or prophylactic treatment of the human body.
The invention also relates to methods for reducing the rate of onset or the severity of Alzheimer""s disease, comprising administering to an animal, such as a human, one or more Group I agents and/or one or more Group II agents in an amount effective to reduce the rate of APP attachment to its receptor.
A further embodiment of the present invention relates to pharmaceutical compositions for therapeutic treatment of Alzheimer""s disease, comprising one or more of Group I agents and/or one or more Group II agents and/or the native receptor molecule of the LDL-receptor family and a pharmaceutically acceptable carrier.
An additional embodiment of the invention relates to the use of LRP for inhibiting the binding of natural ligands to a member of the LDL-receptor family of proteins.
An alternative embodiment of the present invention relates to methods for identifying substances which inhibit the binding of a ligand (RAP) or APP to a protein derived from the LDL-receptor family, comprising the steps of:
a) incubating the receptor, or a soluble form of the receptor, with RAP or APP in the presence of a potential inhibitor substance; and
b) determining the extent of binding of RAP or APP to the receptor or receptor fragment.
Another embodiment of the present invention relates to methods for detecting receptors of the LDL-receptor family, comprising the steps of:
a) incubating a substance derived from a fragment of RAP or APP which contains a binding activity for the receptor with a sample; and
b) determining the extent of binding of the RAP or APP material to the sample.
A further embodiment of the present invention relates to methods for supplying a therapeutically active substance into a carrying cell, characterized in that
a) a fragment of RAP or APP with a binding activity on the LDL-receptor is coupled with the therapeutic substance; and
b) the said material is added to the corresponding cell material, bound to the receptor and in this way the therapeutically active substance is introduced into the cell.
It is to be understood that both the foregoing general description and the following detained description are exemplary and explanatory only and are intended to provide further explanation of the invention as claimed.